Fluidic integrated 3D bioprinting system to sustain cell viability towards larynx fabrication

Abstract Herein, we report the first study to create a three‐dimensional (3D) bioprinted artificial larynx for whole‐laryngeal replacement. Our 3D bio‐printed larynx was generated using extrusion‐based 3D bioprinter with rabbit's chondrocyte‐laden gelatin methacryloyl (GelMA)/glycidyl‐methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post‐printing cell viability, which enabled the generation of a large‐scale cell‐laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio‐larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human‐scale larynxes with in vivo‐like biological functions for laryngectomy patients.

digital models for human tissue and organ regeneration. 1,2 Recent advances in tissue engineering have enabled 3D bioprinting using various biocompatible materials, including living cells, thereby making the product clinically applicable. Human laryngeal allotransplantation has long been anticipated as a therapeutic option for improving the quality of life (breathing without tracheostoma, normal swallowing, and speaking) of laryngectomized patients. 3 However, the requirement for post-transplant immunosuppressive treatment is an ethical concern for the larynx, which is a non-vital organ. Recently, decellularized matrices have also been considered as an alternative option in several studies. [4][5][6] Although the ECM scaffold tends to facilitate tissue regeneration, its preparation method (decellularization approach) can substantially alter the biomechanical properties of the resulting scaffold, which compromises its ability to provide mechanical support during the regeneration process. 6,7 Moreover, the major disadvantage of the allotransplantation/decellularization method is the difficulty in obtaining donor tissues/organs. From this point of view, 3D bioprinting is a potential technology that could solve this problem, despite its technical limitations. Despite significant advances in 3D bio-printing technology, it remains a challenge to print bioinks to achieve long-term stable structures and maintain high cell survival rates after printing.
In this study, we aimed to establish an optimized multimaterial bioprinting methodology for a microextrusion-based 3D bioprinted larynx with chondrocyte-laden GelMA/glycidyl-methacrylated hyaluronic acid (GMHA) bioink. Multimaterial bioprinting is a promising technology integrating multimaterial setups into bioprinting systems for fabricating functional, mechanically stable tissue constructs. 8 For generating biocompatible, multi-cellular, macroscale structure with structural integrity, several studies have suggested various methods, including surface-tension assisted 3D printing, microfluidic systems which facilitate the assembly of 3D tissue models. [9][10][11] However, recent advances in multimaterial bioprinting have limited use in the regeneration of simple anatomical structure. In particular, the field of laryngeal tissue engineering is still in its early stages compared to other organ engineering techniques. This is because the larynx has a very complex structure and various functions compared to other organs. Recently, 3D bioprinting of the larynx using a gelatin methacryloyl (GelMA) bioink blended with decellularized extracellular matrix microparticles has been reported. This method enables the manufacturing of complex laryngeal geometries without requiring support or suspension gels. 12 In our previous study, we developed photocurable GelMA/GMHA hybrid bioink for cartilage regeneration with tonsil-derived mesenchymal stem cells (MSCs). 13 It has been suggested as an optimal bioink with excellent cell viability, mechanical properties, rheological properties, and printability. The success of this study led us to hypothesize that the GelMA/GMHA bioink is a suitable bioink for 3D bioprinting of the human larynx, which is composed of six cartilages (three unpaired and three paired) that form its skeleton. For successful larynx bioprinting, three major strategies were used in this study: (1) mechanical stability was achieved by printing chondrocyte-laden bioinks together with polycaprolactone (PCL); (2) to achieve high cell viability, 200-500 μm sized pores were created in the PCL framework; (3) in particular, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium to the suspension bath during the 3D bioprinting process, thereby improving cell survival during the printing process. We hypothesized that the FS system would decrease cell damage by lowering the temperature of the extruded PCL, and preventing dehydration of the bioink during extended printing time, over 60 min.
To the best of our knowledge, this is the first study to generate a 3D bio-printed artificial larynx using multimaterial bioprinting and evaluate its applicability.

| Rheological and mechanical measurements
The rheological properties (viscosity and shear-stress) of GelMA 7%/ GMHA 5% bioink (G7H5) were measured at 24 C using a rotating rheometer (MCR102, Anton Paar, Ostfildern, Germany) operating in the vibration mode with a strain of 0.1% and frequency of 1 Hz. A total of 1 ml G7H5 bioink (without a photoinitiator, LAP) was placed on a 25-mm plate to measure the viscosity and shear stress. Gelation point test was performed at 30 C. To measure viscoelasticity, 1 ml of G7H5 bioink (with LAP) was placed on a 24-well plate using a 1-cc syringe and cured for 20 s with a UV (365 nm) machine (USHIO, Tokyo, Japan). Subsequently, a disc of diameter 8 mm was created using an 8-mm bipolar punch. For the mechanical test, G7H5 hydrogel, PCL, and PCL + G7H5 tube-shaped constructs with 12-mm outer F I G U R E 1 3D CAD modeling and printing of the rabbit larynx. (a) PCL model and hydrogel model were incorporated into the 3D CAD model of the 3D-bio larynx. (b) Larynx of 20-week-old rabbits was harvested and observed for generating the larynx scaffold. (c) Chondrocytes-laden G7H5 bioink was bio-printed into the space between PCL outer framework. PCL, polycaprolactone; G7H5 bioink, GelMA 7%/GMHA 5% bioink; (1) 11.5 mm; (2) 14.1 mm; (3) 9.2 mm; (4) 8.05 mm; (5) 10.91 mm; (6) 10.95 mm diameter, 10.5-mm inner diameter, and 8-mm height were fabricated.
The wall thickness (300 μm) and pore size (200-500 μm) of the PCL parts were designed to be the same as those of 3D-bio larynx. The compressive strength was measured using a universal testing machine (QM100S, QMESYS, Gunpo, Korea) with a maximum load of 10 kgf at a crosshead speed of 5 mm/min. At least three specimens were used to measure their compressive strengths.

| Preparation of bioink and 3D bioprinting process
A 3D larynx rabbit model was generated using commercial computer- Chondrocytes-loaded G7H5 bioink was prepared as described in a previous study. 13 Briefly, for GelMA, 10% of A-type skin gela- where c represents the glucose concentration (mol/m 3 ) in the G7H5 bioink-based laryngeal model, D d is the dispersion coefficient (m 2 /s), and D e is the diffusion coefficient (m 2 /s). Based on the mass transfer in porous media owing to the concentration gradient, only the diffusion and dispersion phenomena were analyzed in this simulation. In addition, no forced-convection mechanism was considered in the medium domain.
The relationship between diffusion and dispersion is defined as follows: where p denotes the porosity of the porous material, τ f is the effective diffusivity and is equal to p À1=3 , and D f is the fluid diffusion coefficient (m 2 /s). surface, which was not by forced convective flux but by diffusion due to the glucose concentration differences between the 3D-bio larynx and medium domain as follows: where n ! denotes the normal vector, k m is the mass transfer coefficient

| Cell viability by live-and-dead
Cytotoxicity tests were performed using a live/dead assay kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. Images were captured using a K1-fluo confocal laser scanning microscope (Nanoscope Systems, Daejeon, Korea).

| Histological analysis
The specimens were embedded in paraffin blocks and sectioned into

| Statistical analysis
All data are presented as the mean ± standard deviation. Statistical analysis of experimental results was performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). A p value was generated using Student's t-test, with statistical significance set at not significant (ns), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

| Rheological and mechanical properties of the bioink
We recently reported the rheological properties of the GelMA/GMHA bioinks, which were mixed in various ratios at 20, 24, and 30 C. 13 Based on these results, G7H5 bioink was selected in this study, and we examined the rheological properties of the G7H5 bioink ( Figure 4b-e). We also confirmed the compressive strengths of the G7H5 hydrogel, PCL, and PCL + G7H5 hydrogel constructs ( Figure 4f). Our G7H5 bioink exhibited shear-thinning behavior, indicating that the viscosity decreased as the shear rate increased, as shown in Figure 4b. Hydrogels with shear thinning, known as non-T A B L E 1 qRT-PCR primers for cartilage specific gene expression analysis Newtonian fluids, are suitable for extrusion-based bioprinting. 14,15 In this regard, the G7H5 bioink was found to be well-suited for our printing system in this study. The in situ storage modulus (G 0 ) and loss modulus (G 0 ) during UV exposure, in terms of the starting point for gelation, were also confirmed ( Figure 4d)

| Cell proliferation test
The proliferation of chondrocytes gradually increased with time in both groups ( Figure S1). Particularly, G7H5-dissolved media showed higher cell proliferation than the control media (supernatant of DMEM/F12), which indicates that G7H5 bioink promoted more cell growth than DMEM/F12 (free serum, A/A 1%).

| CONCLUSION
Laryngeal cancer is expected to account for 13,150 new cases and 3710 deaths in the United States in 2018. 16 The most common surgical option for advanced laryngeal cancer is total laryngectomy, which negatively impacts the patient's quality of life profoundly, especially actions relating to swallowing, breathing, and speech. 17 Although many approaches are available for laryngeal reconstruction after partial resection, including skin flap, myocutaneous flap, fascial flap, and thyroid gland flap, no satisfactory total laryngectomy replacement is available yet. [16][17][18][19][20] Cells, one of the important elements of 3D bio-printing, are sensitive to environmental changes; therefore, it is important to understand how 3D bio-printing systems affect cells. 21 In general, in situ cell viability is associated with the (1) biocompatibility of the bioink, (2) interaction of cell components with light, (3) thermal stress, and (4) mechanical stress during the printing process. 22 In particular, microextrusion bio-printing, which is used in this study, has some limitations such as low in situ cell viability resulting from shear stress in micro-sized nozzles. 2,4,21,23,24 In addition, a longer printing time causes low post-printing cell viability owing to dehydration of the printed hydrogel. Several studies have suggested the use of biocompatible and mechanically stable macroscale scaffolds. Ragelle et al.
suggested a surface-tension-assisted additive manufacturing method, which employs surface tension forces to coat reticulated supports with cell-laden hydrogel. 9 Yu et al. reported a reconfigurable microfluidic cell-culture system that facilitates the assembly of 3D tissue models by stacking layers containing pre-conditioned microenvironments. 10 Further, the concept called cellular fluidics, suggested by Dudukovic et al., enables the creation of multiscale, cell-based constructs with deterministic structure, porosity, and surface properties; therefore, this method has control over gas-liquid-solid interfaces and fluid flow. 11 In this study, we presented an innovative method for enhancing post-printing cell viability using an FS system. By installing the To achieve structural strength of the printed constructs, we used a PCL outer framework incorporated with pores (200-500 μm). PCL is biocompatible, flexible, and more importantly, has a low melting temperature of 60 C to allow co-printing with cell-laden hydrogel. 25 PCL has been proposed as a tracheal scaffold material in many studies, and our previous studies also confirmed its feasibility as a tracheal substitute because it maintained structural integrity with biocompatibility. 26,27 In this study, G7H5 hydrogel was selected to provide an optimal microenvironment for the printed chondrocytes. In our previous study, G7H5 bioink demonstrated excellent structural stability, including mechanical properties and printability, as well as reliable biocompatibility. 11 In this study, G7H5 bioink was shown to provide a proper microenvironment for the 3D bioprinted chondrocyte. Cell proliferation tests showed that the G7H5 bioink stimulated more chondrocyte proliferation compared to control media. Aggrecan and collagen type II are major components of the extracellular matrix of hyaline cartilage, such as thyroid and cricoid cartilage, 28 and qRT-PCR data in this study demonstrated the promotion of the expression of these genes up to 4 weeks of in vitro culture. SOX-9 is one of the earliest markers expressed in cells undergoing precartilaginous condensation, and RUNX2 is essential for chondrocyte maturation. 29,30 Taken together, these results indicate that the G7H5 bioink provides proper support to maintain chondrocyte innate with proper maturation.
In this study, we confirmed that a computer-generated 3D PCL framework with pores successfully provided structural stability and facilitated nutrient transport to the chondrocyte-laden hydrogel. We used 3D-bio larynx cultured in DMEM/F12 or rabbit omentum for 2 weeks to evaluate in vitro and in vivo cell viability. We observed normal chondrocytes in the G7H5 bioink after 2 weeks of cell printing. These results revealed that the hydrogel parts could be directly connected to the basal media through pores in the PCL parts, and that nutritional support from the media could be sufficiently supplied to the cells in vitro.
To study whether the 3D-bio larynx could replace the defective laryngeal framework, we transplanted 3D-bio larynx into a total laryngectomized rabbit model. In our previous studies, we demonstrated the advantages of the omentum-cultured trachea/esophageal scaffolds: (1) prior implantation of the scaffolds in the omentum is beneficial for revascularization of the scaffolds, (2) therefore, it leads to faster tissue regeneration and reduced associated complications such as scaffold exposure and stenosis at the anastomotic sites. 27,31 A major problem in engineered tissue is the cell death associated with in vivo transplantation. 32 To address this problem, several solutions have been suggested to produce large-scale cell-laden constructs. 2,22,[33][34][35][36][37][38] One possible solution is the pre-vascularization of the scaffold in a bioreactor before transplantation, which can enhance the formation of vasculature within a 3D scaffold. 32,38 As shown in Figure 7c, prior omental implantation led to the formation of homogeneous, vascularized connective tissue on the surface of the 3D-bio larynx.
In conclusion, the FS system can enhance post-printing cell viability, enabling the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient transport. However, our rabbit implantation study did not assess mature tissue regeneration because of the short experimental period. Long-term and large animal studies are required